Melatonin has fascinated the researchers due to its remarkable functional versatility and protective role in several pathophysiological conditions. Indeed, it plays a central role in wide physiological functions, like orchestrating circadian rhythms, along with the regulation of visual, cerebrovascular, reproductive, neuroendocrine, and neuroimmunological functions. Numerous studies have shown melatonin to promulgate a multitude of therapeutic functions that fight sepsis, neurodegenerative disorders, diabetes, biliary and pancreatic malfunctions, hepatotoxicity, cancer, inflammation, and oxidative stress. In addition, it is also a potent antioxidant and free radical scavenger. However, it is also known to regulate the levels of oxidative markers, endogenous antioxidant status, and proinflammatory cytokines. In humans, melatonin (N-acetyl-5-methoxytryptamine; CAS Number: 73-31-4; C13H18N2O2; molecular weight: 232.3) is an endogenous neurohormone secreted primarily from the pineal gland and to a lesser extent by extra pineal tissues such as the retina, harderian gland, and gastrointestinal tract. The non-clinical aspects of pharmacology (primary, secondary and safety pharmacodynamics of endogenous melatonin including in vitro and in vivo studies), pharmacokinetic and toxicokinetic studies of exogenous melatonin in animals are copiously available in the published literature and databank.
While current understanding of endogenous melatonin is substantial, the putative role and mechanisms of endogenous and exogenous melatonin remain unclear. To better understand the scope of the invention it is worth to introduce an overview on basic pharmacokinetic parameters of the drug substance melatonin and to outline its wide spectrum of potential applications and clinical indications, particularly by constant infusion. Melatonin can be administered in a variety of methods well known to those in the art, such as orally, intravenously and topically. Other uncommon uses of melatonin are described in recent publications such as local stimulation of osteointegration of dental implants and prostheses fixed with bone cement.
Melatonin Administered Orally.
When melatonin is administered orally to rats, dogs and monkeys at 10 mg/kg, the absolute bioavailability appeared to be moderate in rats and high in dogs and monkeys [Yeleswaram et al, 1997][1]. Oral administration to human volunteers of up to 3 times 80 mg of melatonin/person in one hour showed that the compound is rapidly absorbed and distributed throughout the human body, the half-life for the first part of the biphasic distribution phase being in the order of minutes. Highest melatonin values were measured in serum, 60 to 150 minutes after oral administration. Elimination of the molecule appeared to be slower than in rodents, since the concentration plateau lasted for several hours. The half-life of the first part of the biphasic elimination phase was found to be 20 to 50 minutes [Waldhauser et al, 1984][2]. When a lower dose of 3 mg of melatonin/volunteer was administered orally, an increase in serum melatonin within 20 minutes after oral administration was reported, followed by a rapid decrease at 240 minutes [Shirakawa et al, 1998][3]. Some authors [Fourtillan et al, 2000][4] reported a terminal half-life of 36 to 45 minutes when melatonin was measured in plasma after oral administration of a dose of 250 μg/person to human volunteers. In literature is reported that maximum blood and saliva concentrations of melatonin were reached 60 minutes after oral uptake of 100 mg of melatonin. The half-life of the molecule in blood was determined as around 41 minutes [Vakkuri et al, 1985][5]. When 2 or 4 mg melatonin/person were administered to human volunteers, only 15% of the ingested dose actually reached the systemic circulation. Most of administered melatonin disappears through the presystemic metabolization [DeMuro et al, 2000][6] and is excreted in urine as sulphatoxy-melatonin, the major conjugation product of 6-hydroxymelatonin. Unchanged melatonin renal clearance was lower than 1% [Fourtillan et al, 2000][4].
Melatonin Administered Intravenously.
Intravenous administration of melatonin to the rat has shown to result in a rapid distribution into plasma and all tissues of the animal, including cerebrospinal fluid and brain [Kopin et al, 1961; Vitte et al. 1988][7][8]. When the active substance was injected at 5 mg/kg, the apparent elimination half-life showed to be 19.8 minutes [Yeleswaram et al, 1997][1]. When melatonin was administered intravenously to the dog and the monkey at 3 mg/kg, the apparent elimination half-lives were measured to be 18.6 and 33.9 minutes respectively [Yeleswaram et al, 1997][1]. When a dose of 23 μg/person of melatonin was administered by intravenous injection to humans, an apparent half-life of 36 to 42 minutes in the systemic circulation was determined [Fourtillan et al, 2000][4].
The toxicokinetics of melatonin in man after intravenous administration are generally characterized by a very short distribution phase in the order of minutes, followed by a steady state concentration in the examined tissues (serum, blood, plasma, brain, etc.) in the order of a couple of hours, and a rapid elimination (half-life of about 40 minutes) after metabolization in the liver [Vitte et al, 1988; Mallo et al, 1990; Le Bars et al, 1991; DeMuro et al, 2000; Fourtillan et al, 2000][8][9][10][6][4]. The major metabolic pathway of melatonin in man has been identified as being the hydroxylation of position 6, followed by conjugation, primarily with sulfate (70%) and, to a smaller extent, with glucuronic acid (6%) [Kopin et al, 1961, Ma et al, 2005][7][12]. Melatonin 6-hydroxylation and O-demethylation have been identified as being mainly CYP1A2 mediated [Facciolá et al, 2001; Ma et al, 2005][11][12].
Melatonin Functions.
Melatonin has several putative functions including circadian rhythms of the body, therefore it is involved in the sleep-wake cycle, functions of the immune and cardiovascular systems, and cell regulation [Reiter et al, 2003; Vijayalaxmi et al, 2002][13][14], regulates the reproductive axis and is a natural antioxidant and potent free radical scavenger [Reiter et al, 2003; Korkmaz et al, 2009; Bonnefont-Rousselot et al, 2010][13][15][16]. Age-related reduction of melatonin has been correlated with disturbance of sleep, deterioration of health and chronic diseases related to oxidative damage, including cancer [Megdal et al, 2005; Reiter et al, 2003][17][13].
Historical review [Wettenberg, 1990][18] dealing with melatonin in humans show heterogeneous relationships between melatonin and other traits and increasing applications as medicament have been proposed to mitigate physiopathological conditions alike:                sleep studies and biological rhythms;        light, retinal sensitivity in humans and the circadian axis;        surgical stress, anaesthesia;        age-related studies (in the human foetus, in children, in elderly and as an antioxidant; the cyclic 3-hydroxymelatonin (3-OHM) biomarker; glucose regulation);        depression and some other psychiatric disorders (fragmented rhythm in schizophrenia, obsessive-compulsive disorder [OCD], fibromyalgic syndrome, appetite-related peptides in ageing);        sleep disturbance in depression, treatment of jet-lag, skin protection from ultraviolet radiation.        
However, during the last decade researchers have continuously encouraged studies suggesting the use of melatonin in the management of serious life threatening conditions. Recent clinical studies (a selection since it would be impossible to list all of them) further suggest that many physiopathological conditions and disorders may benefit from the administration of melatonin, in view of its potent antioxidative effects, such as:                glucose homeostasis [Karamitri et al, 2013][19];        regulate the biliary functions [Glaser et al, 2014][20];        exert important roles on the peripheral reproductive, cellular and organismal (maternal, placental and fetal) physiology [Reiter et al, 2014][21];        ameliorate the physiopathology and influence the behavioural expression of autistic disorder [Tordjman et al, 2013][22];        replace the decreased secretion to treat “sundowning” and other sleep wake disorders typical of Alzheimer Disease (Lin et al, 2013; Wade et al, 2014][23][24];        regulate autophagy and mitophagy [Coto-Montes et al, 2012][25];        correct Parkinson Disease sleep disorders [Suzuki et al, 2011; Downling et al, 2005; Srinivasan et al, 2011][26][27][28];        ameliorate sleep in older Intensive Care Unit (ICU) patients [Sterniczuk et al, 2014][29], improves sleep in Multiple Schlerosis (MS) [Adamczyk-Sowa et al, 2014][30], reduce sleep disturbances in Inflammatory Bowel Disease (IBD) patients [Kinnukan et al, 2013][31].        
The increasing interest in melatonin is reflected in the histogram (FIG. 1) indicating the number of clinical trials per year that have been registered at “ClinicalTrials.gov” (a service of the U.S. National Institutes of Health) from 2004 to the date of offload in 2014. In fact, the growing interest in melatonin therapies in clinical trials being conducted for sepsis [Gitto et al, 2001][32], burns [Sahib et al, 2010][33], ischemic reperfusion [Dominguez-Rodriguez et al, 2007][34], pre-surgical [Caumo et al, 2009; Borazan et al, 2010][35][36], postsurgical [Gitto et al, 2004][37] and perioperative [Jarratt, 2011; Maitra et al, 2013] [38][39], cancer and cancer therapy adjuvant and immunology [Wang et al, 2012; Seely et al, 2011; Ghielmini et al, 1999; Bennukul et al, 2014; Martin et al, 2013; Carrillo-Vico et al, 2013][40][41][42][43][44][45], preeclampsia [Aversa et al, 2012][46], cataract and glaucoma [Ismail et al, 2009][47], radiation protection [Berk et al, 2007][48], perinatal hypoxia and neonatal diseases [Ghitto et al, 2013; Alonso-Alconada et al, 2013][49][50] and in other unexplored therapies similarly requiring an high dosage regimen of melatonin, has not encountered satisfaction in view that a convenient pharmaceutical product of injectable quality melatonin is not yet commercially available. Often melatonin oral form is not an appropriate or usable administration route in critic health conditions. In fact, oral melatonin has high first pass metabolism (>85-90%) in the liver [Lane and Moss, 1985][51], low and variable absolute human bioavailability (average 8.6% female, 16.8% male, range 1-37%) [Fourtillan et al, 2000][4] and high inter-subject dose variability (AUC curve of individual subjects varies by up to 25 times among subjects) [Waldhauser et al, 1984][2], so that constant infusion administration route of melatonin often will be the only viable choice for the deliver and control of an high dosage regimen.
Background of Injectable Preparations.
Patients hospitalized in critical care units with severe sepsis or septic shock, patients undergoing surgery, severe cases of burns and radiation exposure, patients undergoing oncologic therapies, newborn with perinatal or neonatal diseases, and in general patients in critical health conditions may not be able to ingest melatonin via oral route. A skilled person will reasonably recognise that the intravenous infusion of melatonin would represent the unique and most convenient route to treat those patents affected from life threatening diseases, wherein urgent, adequate (massive), accurate and constant dosages of melatonin are required. Hence, a skilled person shall further acknowledge that there is an objective and urgent need to provide the clinicians with pharmaceutical parenteral melatonin to treat those conditions and to extend its use to other unexplored fields of the medicine. Exemplary critical health conditions in humans which would substantially benefit from an infusion of high dosage regimen of injectable melatonin are:
Sepsis
Around 37,000 people die from sepsis in the UK each year, while severe sepsis strikes about 750,000 Americans, and as many as 8 million every year worldwide. It has been estimated that between 28 and 50 percent of these people die, far more than the number of U.S. deaths from prostate cancer, breast cancer and AIDS combined. The number of sepsis cases per year has been on the rise in the United States. Although the Surviving Sepsis Campaign (a performance improvement effort by hospitals across Europe, South America and the United States) has improved outcomes, the mortality rate still remains at 31% overall, and >70% in patients who develop sepsis-induced multiple organ failure. Anyone can get sepsis, but people with weakened immune systems, children, infants, so that frequent are paediatric intensive case patients with sepsis [Bagci et al, 2012][52] and the elderly are most vulnerable, so that sepsis is one of the most common case of death in intensive care units [Moroni et al, 2010; Alan et al, 2010; Srinivasan et al, 2012][53][54][55]. People with chronic illnesses, such as diabetes, AIDS, cancer and kidney or liver disease are also at increased risk, as are those who have experienced a severe burn or physical trauma. It has been recognised by leading clinicians that exogenous antioxidants may be useful in sepsis and more recently the potential for antioxidants acting specifically in mitochondria has been highlighted. In view of the above findings, a group of authors [Galley et al, 2014][56] indicated melatonin as a potential therapy for sepsis and recently undertook a phase I dose escalation study in healthy volunteers to assess the tolerability and pharmacokinetics of 20, 30, 50, and 100 mg oral doses of melatonin capsules. For the phase I trial, oral melatonin was given to five subjects in each dose cohort (n=20). Melatonin was rapidly cleared at all doses with a median (range) elimination half life of 51.7 (29.5-63.2) minutes across all doses. However, there was a considerable variability in maximum melatonin levels within each dose cohort. In view of the high variability among the dose levels obtained following oral administration of capsules manufactured from chemically synthesized melatonin (dose mg/AUC ng/ml/min=20 mg: 1102-13616; 30 mg: 822-2491; 50 mg: 1812-8915; 100 mg: 4458-18229) a skilled person can reasonably conclude that an infusion of melatonin would result the more reliable method of administration for further studies on sepsis in order to assure in all patients the required therapeutic effective dose regimen, especially in paediatric patients or newborns.
Stroke
Each year, approximately 795,000 people suffer a stroke in the United States. About 600,000 of these are first attacks, and 185,000 are recurrent attacks. More than 140,000 people die each year from stroke and is the third leading cause of death. However, stroke is the leading cause of serious, long-term disability. Those figures can be worldwide extended with a certain approximation to other countries. A review paper [Shinozuka et al, 2013][57] supports the approach to deliver melatonin and to target melatonin receptors for neuroprotection in stroke. A number of studies have uniformly reported the important role of melatonin on neuroprotection in animal models of stroke. Experimentally induced stroke is exacerbated in pinealectomized rats [Manev et al, 1996; Kilic et al, 1999][58][59]. Melatonin administration after experimental stroke reduces infarction volume [Pei et al, 2003; Sinha et al, 2001][60][61]. Such a protective effect is seen in both gray and white matter [Lee et al, 2005][62]. Melatonin also reduces inflammatory response [Lee et al, 2007][63], cerebral oedema formation [Kondoh el al, 2009][64], and blood-brain barrier permeability [Chen et al, 2006][65]. Functionally, melatonin administration improves grip strength and motor coordination, and attenuates hyperactivity and anxiety [Kilic et al, 2008][66]. Melatonin secretion is known to decrease age dependently [Brzezinski, 1997][67], suggesting that if melatonin directly affects stroke, then aged people should suffer more strongly from insults of stroke. This may also be ameliorated with melatonin pretreatment. Studies in animal models of stroke have demonstrated that pretreatment of melatonin exerts anti-inflammatory effects and reduces infarction volume [Pei et al, 2002; Pei et al, 2002][68][69]. Numerous studies have documented melatonin-induced neuroprotection against ischemic and hemorrhagic stroke [Borlongan et al, 2000; Reiter et al, 2005][70][71]. In addition, authors describe a novel mechanism of action underlying the therapeutic benefits of stem cell therapy in stroke, implicating the role of melatonin receptors. Experiments warrant consideration to reveal an optimal melatonin treatment strategy that is safe and effective for human application. Neuroprotection shall be achieved in stroke with an higher dosage regimen of melatonin to be promptly and conveniently achieved intravenously by infusion.
Perioperative
Melatonin has some unique properties that are highly desirable in routine peri-operative care so that a new armamentarium of anaesthesiologist has been defined. Available clinical data show that preoperative melatonin is as effective as benzodiazepines in reducing preoperative anxiety with minimal action on psychomotor performance and sleep wake cycle. It may be considered as a safe and effective alternative of benzodiazepines as preoperative anxiolytic. It may have opioid sparing effect, may reduce intraocular pressure, and have role in prevention of postoperative delirium [Generali et al, 2013][72]. The short-term administration of melatonin is free from significant adverse effects also [Maitra et al, 2013][39]. However, the analgesic effects of melatonin have been also evidenced in clinics [Wilhelmsen et al, 2011; Azevedo de Zanette et al, 2014][73][74]. It would appear that patients on melatonin supplement should continue taking them perioperatively because there may be benefits [Jarratt, 2011][38]. It has been also observed that melatonin elicit anaesthesia so that it a suitable tool for patients critical care [Kurdi et al, 2013; Moroni et al, 2010; Alan et al, 2010][75][53][54].
Dialysis
Sleep disorders are common in kidney disease patients on dialysis due to a disturbance in their biological clocks and sleep complaints are common in a dialysis unit. In a survey dialysis patients reported sleep disorders (patients on chronic hemodialysis/HD and continuous peritoneal dialysis/PD) in about 52%. Patients reported trouble falling asleep [Holley et al, 1992][76]. In another survey about 83% patients reported sleep-wake complaints: disturbed sleep (51.8%) secondary to delayed sleep onset (46.5%), frequent night-time awakening (35.2%), restless legs syndrome and generalized (33.3% and 11.1% respectively), 72% admitted to early morning waking and daytime sleepiness (66.7%) [Walker et al, 1995][77]. Another publication confirmed similar sleep disorders in patients on HD and PD [Masaumi et al, 2013][78]. In an earlier publication some authors noted effects of nocturnal hemodialysis on melatonin rhythm (measured in saliva) and sleep-wake behaviour (Koch et al, 2009][79]. in a recent article seventy dialysis patients received melatonin or a placebo for one year. At three months, the previously shown beneficial effect of the short-term use of melatonin on sleep onset was confirmed. The investigators [Russcher et al, 2012][80] also noted improvement of sleep efficiency and sleep time. In contrast, at 12 months none of the measured sleep parameters differed significantly from placebo. However, the researchers observed that the benefits of melatonin on sleep persist over the long term, and that the long-term use of melatonin could improve patients' quality of life. Latter publications also stressed the need to evaluate whether exogenous administration of melatonin can improve the multiple sleep disorders in ESRD (end-stage renal disease) patients [Sharma, 2013][81] and that large randomized controlled trials are needed in order to establish its role in patient population on dialysis [Aperis et al, 2012][82]. In another randomized, double-blind cross-over clinical trial the effectiveness of melatonin versus placebo was tested in patient on conventional daytime HD. The 82 enrolled patients received exogenous melatonin dose set as one tablet at bedtime (3 mg tablet) [Edalat-Nejad et al, 2013][83]. The study suggested that melatonin emerge as a safe therapy for improving sleep quality (SQ) in HD patients. Therefore, in view of the high variance on absorption of oral doses, a skilled person could easily understand benefits and advantages deriving from a convenient parenteral administration melatonin solution admixed to the replacement fluid during dialysis.
Pancreatic Carcinogenesis
Pancreatic cancer has fatal prognosis because of the absence of early symptoms, late diagnosis and the resistance to radio- and chemotherapy. A recent review [Jaworek et al, 2014] [84] refers that in pancreatic carcinoma cell line (PANC-1) melatonin used at high doses affects the Bax/Bcl protein balance, and stimulates the expressions of caspase-9 and caspase-3, thus activating the mitochondrial pathway of apoptosis. Melatonin reduces angiogenesis and decreases proliferation of endothelial cells through inhibition of vascular endothelial factor (VEGF). In animal studies melatonin has been found to increase the efficacy of oncostatic drugs, to reduce the side effects of chemotherapy and to decrease morbidity. These observations suggest that melatonin at high doses could be potentially taken into consideration as the supportive treatment in the therapy of pancreatic cancer, although the effect of melatonin on apoptosis requires further study.
Snake Bite
The results demonstrated that melatonin efficiently alleviated Echis carinatus (EC) venom-induced haemorrhage and myonecrosis. It also mitigated the altered levels of inflammatory mediators and oxidative stress markers of blood components and in liver and kidney homogenates, documented renal and hepatoprotective action of melatonin. The histopathology of skin, muscle, liver and kidney tissues further substantiated the overall protection offered by melatonin against viper bite toxicities. The inability of anti-venoms to block local effects and the fact that melatonin is already a widely used drug promulgating a multitude of therapeutic functionalities, its use in viper bite management is of high interest and should be seriously considered [Katkar el al, 2014][85].
Finally, despite the use of parenteral melatonin in other life-threatening diseases and conditions have not yet been described in literature, authors believe that also patients affected by other serious pathological conditions such as Ebola virus disease and Ebola hemorrhagic fever (EHF) and Dengue and severe Dengue could substantially be alleviated and benefit from the intravenous administration of an high dosage regimen of parenteral melatonin. Authors briefly describe hereby those pathological conditions and the preliminary results of open uncontrolled studies in volunteers.
Ebola (EVD) and Ebola Hemorrhagic Fever (EHF)
Ebola virus disease (EVD), formerly known as Ebola haemorrhagic fever (EHF), is a severe, often fatal illness in humans. EVD outbreaks have a case fatality rate of up to 90%, particularly in aged people. The virus is transmitted to people from wild animals and spreads in the human population through human-to-human transmission. Fruit bats of the Pteropodidae family are considered to be the natural host of the Ebola virus. The incubation period in humans, that is, the time interval from infection with the virus to onset of symptoms, is 2 to 21 days. All people infected show some extent of coagulopathy and impaired circulatory system symptomatology. Bleeding from mucous membranes and puncture sites is reported in 40-50% of cases, while maculopapular rashes are evident in approximately 50% of cases. Sources of bleed include hematemesis, hemoptysis, melena, and aforementioned bleeding from mucous membranes (gastrointestinal tract, nose, vagina and gingiva). However diffuse bleeding (i.e. heavy) is rare; occurrence is usually exclusive to the gastrointestinal tract. In general, development of hemorrhagic symptoms is indicative of a negative prognosis. However, contrary to popular belief, haemorrhage does not lead to hypovolemia and is not the cause of death (total blood loss is low except during labor). Instead, death occurs due to multiple organ dysfunction syndrome (MODS) due to fluid redistribution, hypotension, disseminated intravascular coagulation, and focal tissue necroses. Severely ill patients require intensive supportive care. No licensed specific treatment or vaccine is available for use in people or animals. Nowadays there is a real emergency in several West African countries where outbreaks are not under control of the local health authorities. There is no specific treatment or vaccine for Ebola fever. [WHO/Media centre: Ebola virus disease/Fact sheet n. 103/April 2014]. Authors observed that the hemorrhagic complications (hematemesis, hemoptysis, melena, bleeding from mucous membranes from gastrointestinal tract, nose, vagina and gingiva) could substantially benefit from a coadjuvant treatment with a therapeutically effective amount of intravenous melatonin, feasible with the bulk solution of the invention that make possible such high dosage regimen. The compassionate tests carried out on some patients admitted to an open study showed some favorable and promising outcomes in relation to the obtained benefit to general condition by the concentrated solution of the invention diluted in a saline fluid, preliminary results to be confirmed in a further monitored study.
Dengue and Severe Dengue (Dengue Hemorrhagic Fever/DHF)
The incidence of Dengue has grown dramatically around the world in recent decades. Over 2.5 billion people (over 40% of the world's population) are now at risk from Dengue. There are more than 100 millions new cases of Dengue fever every year throughout the world. Cases across the Americas, South-east Asia and Western Pacific have exceeded 1.2 million cases in 2008 and over 2.3 million in 2010 (based on official data submitted by Member States). Recently the number of reported cases has continued to increase. In 2013, 2.35 million cases of Dengue were reported in the Americas alone, of which 37,687 cases were severe Dengue. An estimated 500,000 people with severe Dengue require hospitalization each year, a large proportion of whom are children. About 2.5% of those affected die. The threat of a possible outbreak of Dengue fever now exists in Europe and local transmission of Dengue was reported for the first time in France and Croatia in 2010 and imported cases were detected in three other European countries. In 2012, an outbreak of Dengue on Madeira islands of Portugal resulted in over 2000 cases and imported cases were detected in 10 other countries in Europe. In 2013, cases have occurred in Florida (United States of America) and Yunnan province of China. Dengue fever is a severe, flu-like illness that affects infants, young children and adults, but seldom causes death. Dengue should be suspected when a high fever (40° C./104° F.) is accompanied by two of the following symptoms: severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands or rash, ecchymosis and petechiae. Symptoms usually last for 2-7 days, after an incubation period of 4-10 days after the bite from an infected mosquito. Severe Dengue is a potentially deadly complication due to plasma leaking, fluid accumulation, respiratory distress, severe bleeding, organ impairment. Warning signs occur 3-7 days after the first symptoms in conjunction with a decrease in temperature (below 38° C./100° F.) and include: severe abdominal pain, persistent vomiting, rapid breathing, bleeding gums, fatigue, restlessness, blood in vomit. The next 24-48 hours of the critical stage can be lethal; proper medical care is needed to avoid complications and risk of death. There is no specific treatment for Dengue fever [WHO/Media centre: Dengue and severe Dengue/Fact sheet n. 117/March 2014; MedlinePlus-NIH/National Institute of Health-USA/October 2012]. Authors observed that Dengue and severe Dengue, in addition to transfusion of fresh blood or platelets to correct the bleeding problems, could substantially benefit from a therapeutically effective infusion of intravenous melatonin at an high dosage regimen, feasible with the bulk solution of melatonin of the instant invention.
Background and Technical State of the Art
Despite the urgent need, medical opinion leaders, clinical doctors and personnel assigned to intensive care units can't afford parenteral preparations of injectable melatonin, since currently there is no commercially available intravenous (IV) dosage form of melatonin.
In fact, melatonin presents many crucial physicochemical aspects that shall be thoroughly considered when attempting to prepare an injectable composition of melatonin, as reported in the publications dealing with this technical challenge. In fact, there are significant technical challenges to overcome by formulating a composition of melatonin to be delivered intravenously. Despite several clinical studies and patents mention the possibility of using melatonin intravenously, the possibility of developing formulations at high concentrations of melatonin is still unsolved since no procedure used for the preparation of high dosages if melatonin, such as for instance at a 10% concentration, has been described in literature. Melatonin is slightly soluble in water (1.2-2.4 mg/ml) [Shida et al, 1994; Kandimalla et al, 1999][86][87], is light sensitive [Andrisano et al, 2000][88], and unstable in aqueous solution [Daya et al, 2001][89]. Many studies have attempted to improve the melatonin solubility including the stability [Dayal et al, 2003; Lee et al, 1997; Lee et al, 1998][90][91][92] but without significant results. Therefore the possibility to prepare a stable solution with an high concentration of melatonin in water looks remote and almost impossible. However, there is evidence that melatonin solution gradually loses potency at all pH values and is not stable when exposed to light or oxygen. Some authors [Daya et al, 2001][89] studied the stability of melatonin solutions over a wide pH range (1.2-12) at room temperature and at 37° C. over a period of 21 days and found that from days 3 to 21 there was a gradual decrease in potency of melatonin throughout this range of pHs, with the decrease not exceeding 30%. The results of the study indicated that solutions of melatonin are relatively stable at room temperature (20° C.) and at 37° C. for at least 2 days. A sterile aqueous solutions of melatonin was prepared at various concentrations (1.0-113.0 micrograms/mil) in pyrogen-free glass vacuum vials and stored at room temperature, 4° C., and at −70° C. for up to 6 months [Cavallo et al, 1995][93]. It was found that the shelf life of melatonin was approximately 5 months at room temperature. The photodegradation products of melatonin were identified as 6-hydroxymelatonin (6-OHM) and N1-acetyl N2-formyl-5-methoxykynuramine (AFMK) and characterized them by NMR, FTIR and mass spectra identified [Andrisano et al, 2000][88]. Both of these compounds also occur endogenously in the body as products of normal hepatic metabolism and radical scavenging, and are not considered toxic. Consequently, many other technical factors shall be carefully considered when designing a stable solution of melatonin especially when an high concentration of melatonin is desired and when additionally the solution is intended for parenteral or intravenous administration to humans.
Two other different intravenous (IV) formulations for melatonin at a strength of 5 mg/ml (0.5%) have been reported in the prior art. The solubility of melatonin in propylene glycol (PG) solution increases slowly until 40% PG and then steeply increases. Solubility of melatonin increased linearly with concentration of 2-hydroxypropyl-beta-cyclodextrin (2-HPBCD/CAS 128446-35-5) without increase in PG. Melatonin solubility in mixtures of PG and 2-HPBCD also increased linearity but was less than the sum of its solubility in 2-HPBCD and PG individually. It was also found that the highest mixture of PG at 40% v/v and 2-HPBCD at 30% w/v had comparable solubility to the other vehicles at much higher concentrations, and had efficiency of melatonin solubilisation [Lee et al, 1997][91]. Melatonin in 10% PG was degraded 85 times more quickly than in aqueous solution without PG at −70° C. On the other hand, the degradation rate constant of melatonin in 2-HPBCD was not changed significantly when compared to water. None of the said solutions gives a satisfactory answer to to necessity to dispose concentrated solutions of melatonin for parental use, offering guarantees of adequate stability in aqueous solution.
EP0835652 (also published as U.S. Pat. No. 5,939,084) describes compositions containing melatonin for both pharmaceutical and cosmetic use, in aqueous solutions of PEG at different concentrations. However, the proposed approach is not without contraindications, since melatonin thus formulated was not stable either in quantitative terms, with loss of content, or in qualitative terms, with the development of degradation products. In addition, due to the presence of isoprene glycol, butylene glycol or propylene glycol in the composition the osmolality of the solutions presented don't have an acceptable level of osmolality for an injectable application or an oral and rectal administration. However, this patent indicates that the composition of one glycol and melatonin shall be a substantially ethanol-free aqueous phase, i.e. which shall not comprise ethanol or traces of ethanol insufficient to dissolve or improve the solubility of melatonin (claim 1).
EP1174134 describes a pharmaceutical or dietary composition for the treatment of cerebral infarction. Said pharmaceutical composition is administered via the oral route, in order to reduce the effects of the infarction. However, this type of administration presents a number of limits, since modest blood concentrations of melatonin are obtained due to its rapid hepatic metabolism. Consequently, low levels of the medicinal product are able to cross the blood-brain barrier and reach the damaged brain areas. Moreover, due to its poor solubility, a significant portion of the dose administered via the oral route is swallowed undissolved in saliva and is responsible for the low and variable bioavailability of melatonin via the gastrointestinal route. The evidence reported that patients with seizures of diverse origin show an alteration of the melatonin rhythm is supportive of its use also for this application.
In another small-scale clinical evaluation it has been found that intravenous (IV) administration of melatonin is appropriate in acute stroke [Cheung et al, 2006][94]. In the preliminary pharmacokinetic and safety study melatonin dissolved in propylene glycol was evaluated in adult male Sprague-Dawley rats, so that it was concluded that melatonin in propylene glycol elevates plasma levels of melatonin with no serious toxicity and that the preparation should be further evaluated in human patients.
Patent publication WO 2012/156565 describes a pharmaceutically acceptable injectable composition comprising water, propylene glycol (PPG) and melatonin, a derivative, a salt, a pro-drug or a solvate of same, which contains no other solvent, co-solvent or dispersing agent. The composition is used as injectable preparation as for instance for intravenous administration. The claimed composition is a mixture of water (from 50% to 95%) and propylene glycol (PPG) (from 5% to 50%). The percentage of PPG may vary from 10% and 30% of the total volume of the composition, while the suitable concentration is comprised between 20 and 30%, while the preferred one contain about 25% of propylene glycol. Melatonin concentration in the water/PPG mixture is variable from 5 to 50 mg/ml; suitable concentrations are between 7 and 20 mg/ml, and the preferred one contains about 10 mg of melatonin/ml of composition (composition expressed as %: melatonin 1.0%; PPG 25%, e.g. 250 mg PPG/mil). Example indicate that vials are sterilized by autoclaving (121° C. during 20 minutes). The stability of vials of the example (melatonin 1.0% solution) has been tested during 14 weeks, and the solution stored at 4° C. presents a small crystallization of the solution.
The recent patent publication WO 2013/068565 describes a powder for reconstitution before use as preparation for injection containing melatonin for the treatment of neonatal cerebral infarction. According to those authors, the invention is achieved by means of a powder for reconstitution dissolved in a mixture of water and polyalkylene glycol (PEG) in which the polyalkylene glycol is present in a quantity from 5 to 40% of the total volume, preferably in a quantity from 10% to 30%, such as to obtain a preparation for injection in the form of a solution containing melatonin. Melatonin composition of Examples from 2 to 13 describe that melatonin is mixed in different proportion with Tween 80® and/or Poloxamer 188® and/or lactose and/or leucine and/or glycine and/or mannitol. The spray-dryer operated at the inlet temperature of 150° C. allows to yield melatonin solid powder particles with certain mean diameter values (limits). The concentration achieved with 1 ml of the preparation of example 10 formulation are: 10.2 mg/ml melatonin (1.02%), 800 mg/ml PEG-400 (80.0%) and 200 mg water (20.0%). No mention is made by authors how powder particles are sterilized and how a endotoxin-free and pyrogen-free injectable preparation is obtained. Patent application further describes that in order to obtain a pharmaceutical form of melatonin that can be used in the treatment of cerebral infarction and in particular in neonatal cerebral infarction, the concentration of melatonin in the pharmaceutical form is from 2 mg/ml to 20 mg/ml (from 0.2 to 2.0%), preferably from 5 mg/ml to 15 mg/ml (from 0.5 to 1.5%) and more preferably from 8 mg/ml to 12 mg/ml (from 0.8 to 1.2%), being the solution also suitable for using in the treatment or prevention of perinatal asphyxia, neonatal cerebral infarction, treatment of sleep disorders in a paediatric patients, treatment of sleep disorders in Autism Spectrum Disorders (ASD) and for use in preanesthesia. Definitely, all prior art uses to dissolve melatonin essentially a binary system (water and PEG), and absent any rational criteria or strategy, other ingredients are also occasionally added, mainly surfactants (Tween 80®, Poloxamer 188®, lecithin or other alike), to yield emulsions, but without increasing the melatonin concentrations, thus remaining unsolved the other technical disadvantages. In fact, when storing the solutions of the prior art at a low temperature (e.g. 4° C.), with the aim to prolong their chemical stability, the ingredients may partially recrystallise with an opalescence or even with precipitations, adding the risk of a loss of active pharmaceutical substance assay and also of administering to the patient an intravenous solution containing suspended particles.
In view of the above results there is a technical evidence that the numerous attempts disclosed in the prior art to provide a stable and convenient pharmaceutical solution with high concentration of melatonin have failed, been either insufficient or inadequate to yield a conveniently stable composition containing an high concentration of melatonin suitable for parenteral administration.